Assay systems for determination of fetal copy number variation

ABSTRACT

The present invention provides processes for determining accurate risk probabilities for chromosome dosage abnormalities. Specifically, the invention provides non-invasive evaluation of genomic variations through chromosome-selective sequencing and non-host fraction data analysis of maternal samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 13/338,963,now U.S. Pat. No. 8,700,338, filed Dec. 28, 2011, which is acontinuation-in-part of U.S. Ser. No. 13/316,154, filed Dec. 9, 2011,which claims priority to U.S. Ser. No. 61/436,135, filed Jan. 25, 2011;this application is also a continuation-in-part of U.S. Ser. No.13/205,570, now U.S. Pat. No. 9,890,421, filed Aug. 8, 2011, which is acontinuation-in-part of U.S. Ser. No. 13/013,732, filed Jan. 25, 2011,which claims priority to U.S. Ser. No. 61/371,605, filed Aug. 6, 2010,all of which are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention provides a non-invasive method for calculating the risk offetal genomic copy number variations such as aneuploidies using maternalsamples including maternal blood, plasma and serum.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and processes will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and processes referenced herein do not constitute prior artunder the applicable statutory provisions.

The American Congress of Obstetricians and Gynecologists (ACOG)recommends that pregnant women be offered non-invasive screening forfetal chromosomal abnormalities. As such existing screening methodsexhibit false positive and negative rates in the range of 5% and 10%respectively, ACOG also recommends that patients categorized byscreening as high risk for fetal aneuploidy be offered invasive testingsuch as amniocentesis or chorionic villus sampling. Although theseinvasive procedures are highly accurate, they are expensive and entail arisk of loss of normal fetus of approximately 0.5-1%. To address theselimitations, non-invasive methods of fetal aneuploidy detection havebeen developed.

In particular, more recent attempts to identify aneuploidies have usedmaternal blood as a starting material. Such efforts have included theuse of cell free DNA (cfDNA) to detect fetal aneuploidy in a sample froma pregnant female, including use of massively parallel shotgunsequencing (MPSS) to quantify precisely the increase in cfDNA fragmentsfrom trisomic chromosomes. The chromosomal contribution resulting fromfetal aneuploidy, however, is directly related to the fraction of fetalcfDNA. Variation of fetal nucleic acid contribution between samples canthus complicate the analysis, as the level of fetal contribution to amaternal sample will vary the amounts needed to be detected forcalculating the risk that a fetal chromosome is aneuploid.

For example, a cfDNA sample containing 4% DNA from a fetus with trisomy21 should exhibit a 2% increase in the dosage of reads from chromosome21 (chr21) as compared to a normal fetus. Distinguishing a trisomy 21from a normal fetus with high confidence using a maternal sample with afetal nucleic acid percentage of 4% requires a large number (>93K) ofchromosome 21 observations, which is challenging and not cost-effectiveusing non-selective techniques such as MPSS.

Thus, improved processes for the calculation of the risk of fetalgenomic copy number variations, e.g., chromosomal contributionabnormalities such as aneuploidies, would be of great benefit in theart.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides methods for evaluating the risk of fetalgenomic copy number variations, including but not limited toaneuploidies. Specifically, the invention provides processes forcalculating risk probabilities to predict the presence or absence of achromosomal abnormality such as a copy number variation or ananeuploidy.

In one general aspect, the invention provides a computer-implementedprocess for determining the presence or absence of a copy numbervariation in a fetal genomic region (e.g., locus or chromosome)comprising the steps of calculating an estimated dosage of a first fetalgenomic region present in a maternal sample, calculating an estimatedcontribution of at least a second fetal genomic region in a maternalsample, and comparing the dosages of the first and second fetal genomicregions to determine the likelihood of a copy number variation in thefirst fetal genomic region.

In some aspects, the copy number variation determined using the methodsof the invention is a chromosomal aneuploidy, and the dosages measurefetal chromosome dosage in a maternal sample. The presence or absence ofa copy number variation of the first fetal genomic region can beestimated by interrogating at least twenty or more polymorphic loci inthe first and second fetal genomic regions, and more preferably byinterrogating at least fifty polymorphic loci in the first and secondfetal genomic regions. The presence or absence of a copy numbervariation can also be estimated by interrogating at least fiveinformative loci in the first and second fetal genomic regions, and morepreferably by interrogating at least twenty informative loci in thefirst and fetal second genomic regions.

In a more specific aspect, the invention provides a computer-implementedprocess to calculate a risk of a fetal aneuploidy comprising estimatingthe dosage of a first fetal chromosome in a maternal sample, estimatingthe dosage of one or more other fetal chromosomes in the maternalsample, calculating a value of the likelihood that a first fetalchromosome is aneuploid by comparing the chromosome dosage of the firstfetal chromosome to the chromosome dosage of the one or more other fetalchromosomes, calculating a value of the likelihood that the first fetalchromosome is disomic by comparing the chromosome dosage of the firstfetal chromosome to the chromosome dosage of the one or more other fetalchromosomes in view of the prior risk of aneuploidy; and calculating arisk of aneuploidy of the first fetal chromosome based on the calculatedvalues of likelihood.

In some aspects, the dosage of one fetal chromosome is compared to thedosage of one or more other individual fetal chromosomes. In otheraspects the dosage of one fetal chromosome can be compared to an averagedosage determined by interrogating two or more other fetal chromosomesand determining an average dosage.

In some aspects, the chromosome dosage of the first chromosome isestimated by interrogating at least twenty polymorphic loci on the firstfetal chromosome, and more preferably by interrogating at least fiftypolymorphic loci on the first fetal chromosome. In other aspects, thechromosome dosage of the first fetal chromosome is estimated byinterrogating at least five informative loci on the first fetalchromosome, more preferably by at least 20 informative loci on the firstfetal chromosome.

The chromosome dosage of the one or more other fetal chromosomes towhich the chromosome dosage of the first fetal chromosome is comparedcan be estimated by interrogating at least five informative loci, ormore preferably at least twenty informative loci, of which all may be ona single chromosome or which may be located on two or more chromosomesdifferent from the first fetal chromosome.

In a specific aspect, the chromosome dosages are calculated for twofetal chromosomes in a maternal sample, and the risk of aneuploidydetermined by a comparison of the chromosome dosages. In this aspect, atleast twenty polymorphic loci are interrogated on each chromosome, andmore preferably at least fifty polymorphic loci are interrogated on eachchromosome. In other aspects, the chromosome dosage of the chromosomesis estimated by interrogating at least five informative loci on eachchromosome, more preferably at least 20 informative loci.

In another general aspect, the invention provides a computer-implementedprocess to calculate a risk of a fetal aneuploidy comprising estimatingthe dosage of a first fetal chromosome in a maternal sample, estimatingthe dosage of one or more other fetal chromosomes in the maternalsample, providing data on prior risk of aneuploidy for at least thefirst fetal chromosome based on extrinsic characteristics, calculating avalue of the likelihood that a first fetal chromosome is aneuploid bycomparing the chromosome dosage of the first fetal chromosome to thechromosome dosage of the one or more other fetal chromosomes in view ofthe prior risk of aneuploidy, calculating a value of the likelihood thatthe first fetal chromosome is disomic by comparing the chromosome dosageof the first fetal chromosome to the chromosome dosage of the one ormore other fetal chromosomes in view of the prior risk of aneuploidy,and calculating a risk of aneuploidy of the first fetal chromosome basedon the calculated values of likelihood.

In some aspects, the invention utilizes a binomial probabilitydistribution to determine the dosages of the different fetal chromosomesin a maternal sample. The binomial probability distribution utilizesfrequency data from informative loci with distinguishing regions thatallow identification and differentiation of nucleic acids from thedifferent sources.

Preferably, the value of the probability of an aneuploidy is calculatedas an odds ratio. In some aspects, when the odds ratio is to determinethe likelihood of a monosomy, the value of the probability of ananeuploidy for the first fetal chromosome can be based on a value of thelikelihood of the chromosome being monosomic and a value of thelikelihood of the chromosome being disomic. In some aspects, the oddsratio is to determine the likelihood of a trisomy, and the value of theprobability of a chromosome dosage abnormality for the first fetalchromosome is based on a value of the likelihood of the chromosome beingtrisomic and a value of the likelihood of the chromosome being disomic.

In some aspects of this embodiment, extrinsic factor(s) are used in theinitial odds ratio calculation, including prior risk data or otherinformation related to gestational age, maternal age, previouspregnancies, and the like. In certain aspects, the data on prior risk ofaneuploidy comprises information related to maternal age. In otheraspects, the data on prior risk of aneuploidy comprises informationrelated to gestational age. In preferred aspects, the data on prior riskcomprises information related to maternal age and gestational age.

Certain aspects of the invention further comprise adjusting an initiallycomputed odds ratio using an extrinsic factor that may affect the oddsratio. Examples of such extrinsic factors include information related tomaternal age, information related to gestational age, informationrelated to previous pregnancies with aneuploid fetus, information onpatient health, information on family history, and the like. Additionalexamples of extrinsic factors include laboratory results, such asPAPP-A, total hCG, beta-free hCG, alpha fetoprotein, unconjugatedestriol and inhibin A, or ultrasound findings such as nuchaltranslucency. In preferred embodiments, the step of adjusting thecomputed odds ratio uses extrinsic factors related to both maternal ageand gestational age.

In some preferred aspects of this embodiment, the maternal sample is acell free maternal sample, and in preferred aspects the cell freematernal sample is maternal blood serum or plasma.

These determinations are a direct comparison of fetal chromosomedosages, and are not dependent on determining an overall dosage of fetalnucleic acids in a maternal sample relative to dosages of maternalchromosomes. It is thus a feature of the invention that only informationon fetal nucleic acid dosage is utilized in the actual calculation ofcopy number variation or aneuploidy

It is a distinguishing feature from other current methodologies that thecopy number variation calculation itself does not require information onmaternal nucleic acid dosage.

In a preferred aspect, the nucleic acid regions used for fetalchromosome dosage calculations of an individual subject are assayed in asingle vessel. In a more preferred aspect, the nucleic acid regionsundergo a universal amplification. In another preferred aspect, thenucleic acid regions are each counted an average of at least 200 times,more preferably at least 300 times, even more preferably 500 times.

It is another feature of the invention that individual fetal chromosomedosages used in the calculations of the invention may be determined in avariety of ways, including determination of polymorphic ratios of fetalchromosomes or the use of binomial probability distributions of thefetal chromosomes in a maternal sample.

DESCRIPTION OF THE FIGURES

FIG. 1 is a block diagram illustrating an exemplary system environment.

DETAILED DESCRIPTION OF THE INVENTION

The processes described herein may employ, unless otherwise indicated,conventional techniques and descriptions of molecular biology (includingrecombinant techniques), genomics, biochemistry, and sequencingtechnology, which are within the skill of those who practice in the art.Such conventional techniques include hybridization and ligation ofoligonucleotides, next generation sequencing, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, New York (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3^(rd) Ed., W. H. Freeman Pub., New York(2000); and Berg et al., Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York (2002), all of which are herein incorporated by reference intheir entirety for all purposes. Before the present compositions,research tools and processes are described, it is to be understood thatthis invention is not limited to the specific processes, compositions,targets and uses described, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present invention, which will be limited only by appendedclaims.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleic acid region” refers to one, more than one, or mixtures of suchregions, and reference to “an assay” includes reference to equivalentsteps and processes known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that eachintervening value between the upper and lower limit of that range—andany other stated or intervening value in that stated range—isencompassed within the invention. Where the stated range includes upperand lower limits, ranges excluding either of those included limits arealso included in the invention.

Unless expressly stated, the terms used herein are intended to have theplain and ordinary meaning as understood by those of ordinary skill inthe art. The following definitions are intended to aid the reader inunderstanding the present invention, but are not intended to vary orotherwise limit the meaning of such terms unless specifically indicated.All publications mentioned herein, and in particular patent applicationsand issued patents, are incorporated by reference for the purpose ofdescribing and disclosing various aspects, details and uses of theprocesses and systems that are described in the publication and whichmight be used in connection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced.

without one or more of these specific details. In other instances,features and procedures well known to those skilled in the art have notbeen described in order to avoid obscuring the invention.

Definitions

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “amplified nucleic acid” is any nucleic acid molecule whoseamount has been increased at least two fold by any nucleic acidamplification or replication process performed in vitro as compared tothe starting amount in a maternal sample.

The term “chromosome dosage” refers to the relative number of copies ofchromosomes in a sample. In the present invention, fetal chromosomedosage for one or more specific chromosomes is determined by comparisonto the chromosome dosage for one or more other fetal chromosomes in amaternal sample. That is, the fetal chromosome dosage calculation in themethods of the invention need not take into consideration maternalchromosome dosage. In one example, if the fetal chromosome dosages are1.0, 1.0, 1.5, 1.0 and 0 for fetal chromosomes 1, 2, 21 and the X and Ychromosomes, respectively, it would appear that the fetus is a female,with a chromosome 21 trisomy. In another example, if the fetalchromosome dosages are 1.0, 1.0, 1.0, 0.5 and 0.5 for fetal chromosomes1, 2, 21 and the X and Y chromosomes, respectively, it would appear thatthe fetus is a male, without a chromosome 21 trisomy. The term“chromosomal dosage abnormality” refers to duplications or deletions ofall (aneuploidy) or part of a chromosome.

The term “diagnostic tool” as used herein refers to any composition orassay of the invention used in combination as, for example, in a systemin order to carry out a diagnostic test or assay on a patient sample.

The term “DNA contribution” refers to the percentage, proportion ormeasurement such as weight by volume of nucleic acid in a sample that iscontributed by a source, such as the mother or a fetus.

The term “extrinsic factor” includes any information pertinent to thecalculation of an odds ratio that is not empirically derived throughdetection of a maternal and fetal locus. Examples of such extrinsicfactors include information related to maternal age, information relatedto gestational age, information related to previous pregnancies with ananeuploid fetus, previous serum screening results, ultrasound findingsand the like. In preferred embodiments, the step of calculating and/oradjusting the computed odds ratio uses extrinsic factors related to bothmaternal age and gestational age.

The term “genomic regions” refers to any genetic region comprising fiveor more informative loci.

The term “hybridization” generally means the reaction by which thepairing of complementary strands of nucleic acid occurs. DNA is usuallydouble-stranded, and when the strands are separated they willre-hybridize under the appropriate conditions. Hybrids can form betweenDNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and along strand containing a region complementary to the short one.Imperfect hybrids can also form, but the more imperfect they are, theless stable they will be (and the less likely to form).

The terms “locus” and “loci” as used herein refer to a nucleic acidregion of known location in a genome.

The term “informative locus” as used herein refers to a locus with oneor more distinguishing regions which is homozygous in one source andheterozygous in another source within a mixed sample.

The term “maternal sample” as used herein refers to any sample takenfrom a pregnant mammal which comprises a maternal source and a fetalsource of nucleic acids (e.g., RNA or DNA).

The term “non-maternal” allele means an allele with a polymorphismand/or mutation that is found in a fetal allele (e.g., an allele with ade novo SNP or mutation) and/or a paternal allele, but which is notfound in the maternal allele.

By “non-polymorphic”, when used with respect to detection of selectednucleic acid regions, is meant a detection of such nucleic acid regionwhich may contain one or more polymorphisms, but in which the detectionis not reliant on detection of the specific polymorphism within theregion. Thus, a selected nucleic acid region may contain a polymorphism,but detection of the region using the assay system of the invention isbased on occurrence of the region rather than the presence or absence ofa particular polymorphism in that region.

As used herein “polymerase chain reaction” or “PCR” refers to atechnique for replicating a specific piece of target DNA in vitro, evenin the presence of excess non-specific DNA. Primers are added to thetarget DNA, where the primers initiate the copying of the target DNAusing nucleotides and, typically, Taq polymerase or the like. By cyclingthe temperature, the target DNA is repetitively denatured and copied. Asingle copy of the target DNA, even if mixed in with other, random DNA,can be amplified to obtain billions of replicates. The polymerase chainreaction can be used to detect and measure very small amounts of DNA andto create customized pieces of DNA. In some instances, linearamplification processes may be used as an alternative to PCR.

The term “polymorphism” as used herein refers to any geneticcharacteristic in a locus that may be indicative of that particularlocus, including but not limited to single nucleotide polymorphisms(SNPs), methylation differences, short tandem repeats (STRs), and thelike.

The term “polymorphic locus” as used herein refers to a locus with twoor more detectable alleles within a population. Generally, a polymorphiclocus will have the most common allele less than 70%.

Generally, a “primer” is an oligonucleotide used to, e.g., prime DNAextension, ligation and/or synthesis, such as in the synthesis step ofthe polymerase chain reaction or in the primer extension techniques usedin certain sequencing reactions. A primer may also be used inhybridization techniques as a means to provide complementarity of anucleic acid region to a capture oligonucleotide for detection of aspecific nucleic acid region.

The term “research tool” as used herein refers to any composition orassay of the invention used for scientific enquiry, academic orcommercial in nature, including the development of pharmaceutical and/orbiological therapeutics. The research tools of the invention are notintended to be therapeutic or to be subject to regulatory approval;rather, the research tools of the invention are intended to facilitateresearch and aid in such development activities, including anyactivities performed with the intention to produce information tosupport a regulatory submission.

The term “selected nucleic acid region” as used herein refers to anucleic acid region corresponding to a genomic region on an individualchromosome. Such selected nucleic acid regions may be directly isolatedand enriched from the sample for detection, e.g., based on hybridizationand/or other sequence-based techniques, or they may be amplified usingthe sample as a template prior to detection of the sequence. Nucleicacids regions for use in the processing systems of the present inventionmay be selected on the basis of DNA level variation between individuals,based upon specificity for a particular chromosome, based on CG contentand/or required amplification conditions of the selected nucleic acidregions, or other characteristics that will be apparent to one skilledin the art upon reading the present disclosure.

The terms “sequencing”, “sequence determination” and the like as usedherein refers generally to any and all biochemical processes that may beused to determine the order of nucleotide bases in a nucleic acid.

The term “specifically binds”, “specific binding” and the like as usedherein, refers to one or more molecules (e.g., a nucleic acid probe orprimer, antibody, etc.) that binds to another molecule, resulting in thegeneration of a statistically significant positive signal underdesignated assay conditions. Typically the interaction will subsequentlyresult in a detectable signal that is at least twice the standarddeviation of any signal generated as a result of undesired interactions(background).

The term “value of the likelihood” refers to any value achieved bydirectly calculating likelihood or any value that can be correlated toor otherwise indicative of a likelihood.

The term “value of the probability” refers to any value achieved bydirectly calculating probability or any value that can be correlated toor otherwise indicative of a probability.

The Invention in General

The present invention provides processes for determining the likelihoodof a fetal copy number variation or an aneuploid chromosome in a fetusby directly comparing the level of fetal chromosome dosages from atleast two fetal chromosomes without using maternal chromosome dosage asa direct comparator in the determination. Fetal DNA contribution to thematernal sample can be determined using various methods that distinguishthe fetal nucleic acids from corresponding maternal nucleic acids. Oncefetal DNA contribution in the maternal sample is determined, the dosageof specific fetal genomic regions (including chromosomes) can becompared to the dosages for other fetal chromosomes in the sample toidentify any statistical differences that would indicate that one ormore fetal genomic regions has a variation, e.g., a copy numbervariation or an aneuploidy. That is, the risk of fetal copy numbervariation or aneuploidy is determined by looking at only fetalchromosome dosage, without using maternal chromosome or locus dosagedeterminations.

Determination of Fetal DNA Contribution in a Maternal Sample

The fetal DNA contribution in a maternal sample is used as a part of therisk calculation of the present invention.

The fetal DNA contribution in the maternal sample used in the odds riskcalculation can be estimated using a variety of techniques. Theprocesses for detection include various strategies including but notlimited to those described herein. One of skill in the art willrecognize that any method by which one can estimate the contribution ofa fetal DNA in a maternal sample can be used in determination of thefetal chromosome dosage, which in turn is used in the calculation ofaneuploidy.

In general, the fetal DNA contribution can be determined relative to theoverall DNA levels in a maternal sample, and the fetal DNA contributioncan be used to identify genomic regions which are either overrepresented(as in the case on an extra copy of a genomic region) orunderrepresented (as in the case of a missing copy of a genomic region).

In some aspects, fetal DNA contribution in the maternal sample canprovide important information on the expected statistical presence ofchromosomal dosage. Variation from the expected statistical presence maybe indicative of fetal aneuploidy, and in particular a fetal trisomy ormonosomy of a particular chromosome.

In certain aspects, determination of fetal polymorphisms requirestargeted SNP and/or mutation analysis to identify the presence of fetalDNA in a maternal sample. In some aspects, prior genotyping of thefather and/or mother may be used. For example, the parents may haveundergone genetic screening to identify disease markers, e.g., markersfor disorders such as cystic fibrosis, muscular dystrophy, spinalmuscular atrophy or even the status of the RhD gene. Differences inpolymorphisms, copy number variants or mutations between fetal andmaternal nucleic acids can be used to determine the fetal DNAcontribution in a maternal sample.

In one preferred aspect, the percent or proportion of fetal cell freeDNA in a maternal sample can be quantified using multiplexed SNPdetection without prior knowledge of the maternal or paternal genotype.In this aspect, two or more selected polymorphic nucleic acid regionswith a known SNP in each region are used. In a preferred aspect, theselected polymorphic nucleic acid regions are located on an autosomalchromosome that is unlikely to be aneuploid, e.g., not chromosomes 21,18, or 13. The selected polymorphic nucleic acid regions from thematernal sample (e.g., plasma) are amplified. In a preferred aspect, theamplification is universal; and in a preferred embodiment, the selectedpolymorphic nucleic acid regions are amplified in one reaction in onevessel. Each allele of the selected polymorphic nucleic acid regions inthe maternal sample is determined and quantified. In a preferred aspect,high throughput sequencing is used for such determination andquantification.

Loci are thus identified where the maternal and fetal genotypes aredifferent; e.g., the maternal genotype is homozygous and the fetalgenotype is heterozygous. For example, identification of informativeloci can be accomplished by observing a high frequency of one allele(>80%) and a low frequency (<20% and >0.15%) of the other allele for aparticular selected nucleic acid region. The use of multiple loci isparticularly advantageous as it reduces the amount of variation in themeasurement of the abundance of the alleles between loci. All or asubset of the loci that meet this requirement within a genomic region ofinterest can be used to determine fetal DNA contribution in the maternalsample using statistical analysis, as described in more detail herein.In one aspect, fetal DNA contribution in the maternal sample isdetermined by summing the low frequency alleles from two or more locitogether, dividing by the sum of the low and high frequency alleles andmultiplying by two.

In one preferred embodiment, the present invention utilizes allelicinformation where there is a distinguishable difference between thefetal and maternal DNA (e.g., a fetal allele containing at least oneallele that differs from the maternal allele) in determination of fetalDNA contribution in the maternal sample. Data pertaining to allelicregions that are the same for maternal and fetal DNA are thus notselected for analysis, or are removed from the pertinent data prior todetermination of the fetal DNA contribution in the maternal sample so asnot to mask the useful data. Additional exemplary processes forquantifying fetal DNA contribution in maternal plasma can be found,e.g., in Chu, et al., Prenat. Diagn., 30:1226-29 (2010), which isincorporated herein by reference.

In a related aspect, data from selected nucleic acid regions may beexcluded from the calculation of fetal DNA contribution if the data fromthe region appears to be an outlier due to experimental error or fromidiopathic genetic bias within a particular sample. In another aspect,selected data from certain nucleic acid regions may undergo statisticalor mathematical adjustment such as normalization, standardization,clustering, or transformation prior to summation or averaging. Inanother aspect, data from selected nucleic acid regions may undergo bothnormalization and data experimental error exclusion prior to summationor averaging. The normalization may be performed for each of the dosagescompared to determine the aneuploidy, or the normalization may beperformed for one or a subset of the dosages compared to determine theaneuploidy.

Determination of Fetal DNA Contribution in a Maternal Sample UsingEpigenetic Allelic Ratios.

Certain genes have been identified as having epigenetic differencesbetween the fetus and the mother, and such genes are candidate loci forfetal DNA markers in a maternal sample. See, e.g., Chim S S, et al.,PNAS USA, 102:14753-58 (2005). These loci, which are unmethylated in thefetus but are methylated in maternal blood cells, can be readilydetected in maternal plasma. The epigenetic allelic ratio for one ormore of such sequences known to be differentially-methylated in fetalDNA as compared to maternal DNA can be determined for a genomic region(e.g., a chromosome). The comparison of methylated and unmethylatedamplification products from a maternal sample can then be used toquantify fetal DNA contribution in the maternal sample.

To determine methylation status of nucleic acids in a maternal sample,the nucleic acids of the sample are subjected to bisulfite conversion.Conventional processes for such bisulphite conversion include, but arenot limited to, use of commercially available kits such as theMethylamp™ DNA Modification Kit (Epigentek, Brooklyn, N.Y.). Allelicfrequencies and ratios can be directly calculated and exported from thedata to determine the dosage of fetal genomic regions in the maternalsample.

Determination of Fetal Chromosome Dosage

When measuring chromosome or locus dosage, the fetal loci used tocalculate chromosome dosage can be selected from a maternal sample priorto detection, i.e. selectively isolated from a maternal sample prior todetection using amplification or capture techniques such ashybridization. Alternatively, the fetal loci used in estimation ofchromosome dosage may be selected after detection, e.g., by filteringfrequency data generated from techniques such as massively parallelshotgun sequencing of nucleic acids within the maternal sample.

In some specific aspects, estimation of chromosome dosage employshighly-multiplexed sequencing of selected loci from specific chromosomesof interest. Chromosome-selective sequencing can be used to assaynumerous loci simultaneously in a single reaction, enabling estimationof both fetal chromosome dosage of fetal DNA contribution in thematernal sample. Subsequently, a novel risk calculation of the inventioncan employed, which leverages chromosome dosage and fetal DNAcontribution estimates to compute the likelihood of chromosomal dosageabnormalities (e.g., fetal trisomy) in each subject.

In a preferred example, the chromosome dosage for a fetal chromosome isdetermined on a chromosome-by-chromosome basis. For instance, frequencyinformation for fetal chromosome 21 can be compared to fetal chromosome18. In another example, the combined dosage of two or more chromosomescan be used as a comparator for determining an aneuploidy in a singlechromosome, e.g., the chromosome dosages of chromosomes 1 and 2 can beused as a comparator for identifying the presence or absence of ananeuploidy in chromosome 21. In certain aspects, the chromosome used asa comparator for one chromosome may also be a chromosome interrogatedfor possible abnormalities, e.g., the chromosome dosage of chromosome 18may be compared to the chromosome dosage of chromosome 21 to identifythe presence or absence of an aneuploidy in either chromosome. Inanother aspect, the chromosome(s) used as a comparator specifically isnot a chromosome interrogated for possible dosage abnormalities.

Determining which genetic loci are contributed to the fetus fromnon-maternal sources allows the estimation of fetal genomic regiondosage (e.g., chromosome dosage) in a maternal sample, and thus providesinformation used to calculate statistically significant differences inthe dosages for genomic regions (e.g., chromosomes) of interest.

In a general aspect, data from 20 or more polymorphic loci are used foranalysis of fetal chromosome dosage. In another preferred aspect, datafrom 30 or more polymorphic loci are used for the analysis. In anotherpreferred aspect, data from 40 or more polymorphic loci are used for theanalysis. In another preferred aspect, data from 50 or more loci areused for the analysis. In another preferred aspect, data from 100 ormore loci are used for the analysis. In another preferred aspect, datafrom 200 or more loci are used for the analysis.

In a preferred aspect, data from 5 or more informative loci are used forthe analysis of fetal chromosome dosage. In another preferred aspect,data from 20 or more informative loci are used for the analysis. Inanother preferred aspect, data from 40 or more informative loci are usedfor the analysis. In another preferred aspect, data from 50 or moreinformative loci are used for the analysis. In another preferred aspect,data from 100 or more informative loci are used for the analysis. Inanother preferred aspect, data from 200 or more informative loci areused for the analysis.

In another aspect, one or more indices are used to identify the sample,the locus, the allele or the identification of the nucleic acid. Suchindices are as described in co-pending application Ser. Nos. 13/205,490and 13/205,570 hereby incorporated herein by reference in theirentirety.

In one preferred aspect, fetal chromosome dosage is quantified usingtandem SNP detection in the maternal and fetal alleles. Techniques foridentifying tandem SNPs in DNA extracted from a maternal sample aredisclosed in Mitchell et al, U.S. Pat. No. 7,799,531 and U.S. patentapplication Ser. Nos. 12/581,070, 12/581,083, 12/689,924, and12/850,588. These references describe the differentiation of fetal andmaternal loci through detection of at least one tandem single nucleotidepolymorphism (SNP) in a maternal sample that has a different haplotypebetween the fetal and maternal genome. Identification and quantificationof these haplotypes can be performed directly on the maternal sample andused to determine the fetal frequency of genomic regions, includingfetal chromosome dosage, in the maternal sample.

As described in relation to calculation of fetal DNA contributionpreviously, data from selected nucleic acid regions may be excluded fromthe calculation of fetal chromosome dosage if the data from the regionappears to be an outlier due to experimental error or from idiopathicgenetic bias within a particular sample. In another aspect, selecteddata from certain nucleic acid regions may undergo statistical ormathematical adjustment such as normalization, standardization,clustering, or transformation prior to summation or averaging. Inanother aspect, data from selected nucleic acid regions may undergo bothnormalization and data experimental error exclusion prior to summationor averaging. The normalization may be performed for each of the dosagescompared to determine the aneuploidy, or the normalization may beperformed for one or a subset of the dosages compared to determine theaneuploidy.

Empirical Techniques for the Estimation of Chromosome Dosage

Fetal chromosome dosage can be estimated using various differenttechniques, as will become apparent to one skilled in the art uponreading the present disclosure. Preferably, the techniques used involvedetermination of sequence differences between maternal and non-maternalsequences. This can be accomplished using array-based hybridizationprocesses, such as those described in U.S. Pat. Pub. No. 2011/0172111.In other aspects, the biomolecules are detected using nanoporetechnology detection, such as those described in U.S. Pat. Pub. No.2011/0124518. In preferred embodiments, the techniques used involvesequence determination of all or a portion of the fetal genomic regionsused in the dosage calculations of the invention.

In certain aspects, the nucleic acids are sequenced and compared usingpolymorphisms that differentiate between maternal and fetal alleles in asample, using methods such as those described in U.S. Pat. Nos.7,727,720, 7,718,370, 7,598,060, 7,442,506, 7,332,277, 7,208, 274, and6,977,162. Briefly, the methods utilize polymorphic detection toidentify chromosomal abnormalities. Sequences are determined at allelesthat are homozygous in the mother and heterozygous in the fetus, and aratio for the heterozygous alleles are determined. The ratio for theheterozygous alleles is used to indicate the presence or absence of achromosomal abnormality.

In yet another aspect, estimation of chromosomal dosage abnormalitiesutilizes sequence identification of tandem polymorphisms, such as thatdescribed in, e.g., U.S. Pat. No. 7,799,531, and U.S. Pub. Nos.2011/0117548, 2011/0059451, 2010/0184044, 2010/184043, and 2008/0020390.Briefly, tandem SNPs are detected and used to differentiate maternal andfetal alleles in a maternal sample to allow calculation of fetalchromosome dosages, thereby identifying fetal chromosomal abnormalities.

In a preferred aspect, the estimation of fetal chromosomal dosageutilizes selected amplification and sequence detection of representativeloci. Such techniques are disclosed in, e.g., U.S. application Ser. Nos.13/013,732, 13/205,490, 13/205,570, and 13/205,603, all of which areincorporated herein in their entirety. These techniques utilizedetection of genomic regions using fixed sequence oligonucleotides andjoining the fixed sequence oligonucleotides via ligation and/orextension. This can be accomplished using a combination of ligation andamplification, e.g., the ligation of two or more fixed sequenceoligonucleotides and optionally a bridging oligonucleotide that iscomplementary to a region between the fixed sequence oligonucleotides.In another example, this can be accomplished using a combination ofextension, ligation and amplification. In a preferred example theamplification is a universal amplification. Preferably, theamplification occurs in one vessel. Numerous methods of sequencedetermination are compatible with the assay systems of the inventions.Exemplary methods for sequence determination include, but are notlimited to, hybridization-based methods, such as disclosed in Drmanac,U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al,U.S. patent publication 2005/0191656, which are incorporated byreference, sequencing by synthesis methods, e.g., Nyren et al, U.S. Pat.Nos. 7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos.7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc.Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing asdescribed in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311,6,828,100, and 6,210,891; and ligation-based sequencing determinationmethods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, andChurch et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

Sequence information of fetal loci may be determined using methods thatdetermine many (typically thousands to billions) of nucleic acidsequences in an intrinsically parallel manner, where many sequences areread out preferably in parallel using a high throughput serial process.Such methods include but are not limited to pyrosequencing (for example,as commercialized by 454 Life Sciences, Inc., Branford, Conn.);sequencing by ligation (for example, as commercialized in the SOLiD™technology, Life Technology, Inc., Carlsbad, Calif.); sequencing bysynthesis using modified nucleotides (such as commercialized in TruSeq™and HiSeq™ technology by Illumina, Inc., San Diego, Calif., HeliScope™by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS byPacific Biosciences of California, Inc., Menlo Park, Calif.), sequencingby ion detection technologies (Ion Torrent, Inc., South San Francisco,Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., MountainView, Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and likehighly parallelized sequencing methods.

Alternatively, in another aspect, the entire length of the amplificationproduct or a portion of the amplification product may be analyzed usinghybridization techniques. Methods for conducting polynucleotidehybridization assays for analyzing nucleic acids have been welldeveloped in the art. Hybridization assay procedures and conditions willvary depending on the application and are selected in accordance withthe general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred aspects. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

Reference Sets for Calculating the Risk of Fetal Aneuploidy

In certain aspects of the invention, reference samples comprising one ormore maternal samples with known levels of fetal chromosome dosages frompatients carrying normal and/or abnormal (e.g., trisomic) fetuses can beused to identify risk of an aneuploidy. The fetal chromosome dosagespresent in one or more reference samples can be directly compared tofetal chromosome dosages in a test maternal sample to identify the riskof aneuploidy for a particular fetal chromosome. For example,chromosomal dosage estimations and variations can be calculated bycomparing a test maternal sample to a reference maternal sample that hasa corresponding level of fetal DNA contribution in the sample. As wellas being matched based on overall fetal DNA contribution, referencematernal samples may be selected for comparison based on characteristicssuch as corresponding maternal age, corresponding gestational age, andthe like.

These reference maternal samples can thus be used as a comparator forfetal chromosome dosage to identify the risk of an aneuploidy in a testmaternal sample. Preferably, a test sample can be compared to one ormore reference maternal samples from subjects carrying a diploid fetusand reference maternal samples from one or more subjects carrying afetus that has an aneuploidy. The comparison of the test maternal sampleto both reference samples can be used to calculate a value of thelikelihood that the test maternal sample is from a subject carrying ananeuploidy fetus. The reference sample(s) may be tested in the samevessel or reaction as the maternal sample being tested. Differentiationof the test maternal sample results from the reference sample resultscould be accomplished by interrogating different loci in the referencesample(s) than the loci in the maternal sample being tested.Differentiation of the test maternal sample results also could beaccomplished by interrogating the same loci in the reference sample(s)and test sample, but where there is a difference in sequence between thereference sample(s) versus the maternal sample being tested. Thereference sample(s) may be synthesized or engineered to allow for thesechanges in sequence to be made. One likely advantage of having thereference sample(s) being in the same vessel or reaction as the maternalsample being tested is any assay variance would likely impact both thetest and reference samples.

In one example, reference samples can be used as “normal” comparatorsfor identifying the risk of a fetal aneuploidy. The fetal dosage of agenomic region (e.g., a chromosome of interest) in a test maternalsample can be compared to one or more appropriate corresponding normalreference samples that have approximately the same overall fetal DNAcontribution in the reference sample as that found in the test sample.Test maternal samples with fetal chromosome or loci contribution(s) thatfall outside normal range, as determined using the reference sample orsamples, are identified as at risk of aneuploidy.

In another example, reference samples can be used as “affected”comparators for identifying the risk of a fetal aneuploidy. The dosageof one or more fetal genomic regions (e.g., a chromosome of interest) ina test maternal sample can be compared to one or more reference samplesthat have approximately the same overall fetal DNA contribution as thetest sample and a known copy number variation, e.g., a trisomy. Testmaternal samples that demonstrate a chromosome or locus contributionthat falls within a range that corresponds to the known affectedreference sample or samples, are identified as at risk of aneuploidy.

In some embodiments, chromosome dosages of a maternal test sample arecompared to two or more reference samples that have a range of fetal DNAcontribution relevant to the test maternal sample. In certain aspects,the range of fetal DNA contribution includes a range of two percentagepoints (e.g., 10-11%). In other aspects, the range of fetal DNAcontribution includes a range of three percentage points (e.g., 8-10%).In yet other aspects, the range of fetal DNA contribution includes arange of four percentage points (e.g., 4-7%). In yet other aspects, therange of fetal DNA contribution includes a range of five percentagepoints (e.g., 5-9%).

In a more specific example, chromosome dosages of a specific fetalchromosome (e.g., chromosome 21) in a test maternal sample that has anempirically determined fetal DNA contribution of 5% can be compared tothe fetal chromosome dosage of the same chromosome in a referencematernal sample that has also been determined to have an overall fetalDNA contribution of 5% and which is known to be from a subject carryinga diploid fetus. Alternatively, fetal chromosome dosages of a specificchromosome (e.g., chromosome 21) in a test maternal sample that has anempirically determined fetal DNA contribution of 5% can be compared tothe chromosome dosage of two or more reference maternal samples thathave been determined to have an overall fetal DNA contribution within anidentified range, e.g., samples with fetal DNA contribution between 4-6%known to be from subjects carrying a diploid fetus. Test maternalsamples with fetal chromosome dosages statistically greater than thedetermined range of normal defined by the reference samples would beidentified as having an elevated risk of trisomy. Test maternal sampleswith chromosome dosages statistically lower than the determined range ofnormal defined by the reference samples would be identified as having anelevated risk of monosomy.

In another example, chromosome dosages of a specific fetal chromosome(e.g., chromosome 21) in a test maternal sample that has an empiricallydetermined fetal DNA contribution of 7% can be compared to thechromosome dosage of that chromosome in a reference maternal sample thathas also been determined to have an overall fetal DNA contribution of 7%and which is known to be from a subject carrying a fetus with ananeuploidy (e.g., trisomy 21). Alternatively, fetal chromosome dosagesof a specific chromosome (e.g., chromosome 21) in a test maternal samplethat has an empirically determined fetal DNA contribution of 7% can becompared to the chromosome dosage of two or more reference maternalsamples that have been determined to have an overall fetal DNAcontribution within an identified range, e.g., samples with fetal DNAcontribution between 5-9% known to be from subjects carrying a fetuswith an aneuploidy (e.g., trisomy 21). Test maternal samples with fetalchromosome dosages statistically greater than the determined range ofnormal defined by the reference samples would be identified as having anelevated risk of trisomy. Test maternal samples with fetal chromosomedosages statistically lower than the determined range of normal definedby the reference samples would be identified as having an elevated riskof monosomy.

Multiple reference samples can form a reference set that can be used ascomparators for multiple test maternal samples with varying, specificcharacteristics, and thus would be useful as comparators for widerpopulations of patients. Such a reference sample set would preferablyinclude reference samples that represent different ranges of fetal DNAcontribution, as well as different dosage frequencies for one or morechromosomes or loci of interest. Such reference sample sets may becreated using 2 or more, or preferably 5 or more samples from subjectswith a diploid fetus, where the different samples in the reference sethave different levels of fetal DNA contribution. In addition, referencesets may be further refined by sample characteristics such ascorresponding maternal age, corresponding gestational age, and the like.

In a preferred aspect, the loci selected for analysis in the maternaltest sample include in a single reaction both loci for determination offetal DNA contribution as well as loci of interest corresponding to oneor more chromosome(s) or one or more portion(s) of a chromosome ofinterest for determination of dosage. Use of a single reaction helps tominimize the risk of contamination or bias that may be introduced usingseparate reactions, which may otherwise skew results. In fact, themethods of the present invention are preferably performed as multiplexedor even highly-multiplexed reactions, where loci for determining fetalDNA contribution and chromosome dosage are interrogated in a singlereaction for each sample. In preferred embodiments, the multiplexingassays described in U.S. application Ser. Nos. 13/013,732, 13/205,490,13/205,570, and 13/205,603 are used, as these assays query bothpolymorphic and non-polymorphic loci in a maternal sample in a singlemultiplexed reaction.

In addition to the methods described earlier, the variation in the assaymay be reduced when all of the nucleic acid regions for each sample areinterrogated in a single reaction in a single vessel. Similarly, thevariation in the assay may be reduced when a universal amplificationsystem is used. Furthermore, the variation of the assay may be reducedwhen the number of cycles of amplification is limited.

Universal Amplification

In preferred aspects of the invention, the nucleic acid loci arepreferably amplified in a multiplexed assay system. This is preferablydone through use of universal amplification of the various loci to beanalyzed using the assay systems of the invention. Universal primersequences are added to the amplification products either during orfollowing selective amplification of loci of interest, so that the locimay be further amplified in a single universal amplification reaction.For example, universal primer sequences may be added to the during theselective amplification process, i.e., the primers for selectiveamplification have universal primer sequences that flank a locus.Alternatively, adapters comprising universal amplification sequences canbe added to the ends of the loci selected for amplification as adaptersfollowing amplification and isolation of the selected nucleic acids fromthe mixed sample.

In one exemplary aspect, nucleic acids are initially amplified from amaternal sample using primers comprising a region complementary toselected regions or loci of the chromosomes of interest and universalpriming sites. The initial selective amplification is followed by auniversal amplification step to increase the number of nucleic acidregions for analysis. This introduction of primer regions to the initialamplification products allows a subsequent controlled universalamplification of all or a portion of selected nucleic acids prior to orduring analysis, e.g., sequence determination.

Bias and variability can be introduced during DNA amplification, such asthat seen during polymerase chain reaction (PCR). In cases where anamplification reaction is multiplexed, there is the potential that lociwill amplify at different rates or efficiency. Part of this may be dueto the variety of primers in a multiplex reaction with some havingbetter efficiency (i.e. hybridization) than others, or some workingbetter in specific experimental conditions due to the base composition.Each set of primers for a given locus may behave differently based onsequence context of the primer and template DNA, buffer conditions, andother conditions. A universal DNA amplification for a multiplexed assaysystem will generally introduce less bias and variability.

Accordingly, in a preferred aspect, a small number (e.g., 1-10,preferably 3-5) of cycles of selective amplification or nucleic acidenrichment in a multiplexed mixture reaction are performed, followed byuniversal amplification using introduced universal priming sites. Thenumber of cycles using universal primers will vary, but will preferablybe at least 10 cycles, more preferably at least 15 cycles, even morepreferably 20 cycles or more. By moving to universal amplificationfollowing one or a few selective amplification cycles, the bias ofhaving certain loci amplify at greater rates than others is reduced.

Optionally, the assay system will include a step between the selectiveamplification and universal amplification to remove any excess nucleicacids that are not specifically amplified in the selectiveamplification.

The whole product or an aliquot of the product from the selectiveamplification may be used for the universal amplification. The same ordifferent conditions (e.g., polymerase, buffers, and the like) may beused in the amplification steps, e.g., to ensure that bias andvariability is not inadvertently introduced due to experimentalconditions. In addition, variations in primer concentrations may be usedto effectively limit the number of sequence specific amplificationcycles.

In certain aspects, the universal primer regions of the primers oradapters used in the assay system are designed to be compatible withconventional multiplexed assay methods that utilize general primingmechanisms to analyze large numbers of nucleic acids simultaneously inone reaction in one vessel. Such “universal” priming methods allow forefficient, high volume analysis of the quantity of nucleic acid regionspresent in a mixed sample, and allow for comprehensive quantification ofthe presence of nucleic acid regions within such a mixed sample for thedetermination of aneuploidy.

Examples of such assay methods include, but are not limited to,multiplexing methods used to amplify and/or genotype a variety ofsamples simultaneously, such as those described in Oliphant et al., U.S.Pat. No. 7,582,420, which is incorporated herein by reference.

Some aspects utilize coupled reaction for multiplex detection of nucleicacid sequences where oligonucleotides from an early phase of eachprocess contain sequences which maybe used in processes used in one ormore later phases of the method. Exemplary processes for amplifyingand/or detecting nucleic acid in samples can be used or alone incombination, including but not limited to the methods described below,each of which are incorporated by reference in their entity for purposesof teaching various elements that can be used in the assay systems ofthe invention.

In Certain aspects, the assay system of the invention utilizes one ofthe following combined selective and universal amplification techniques:(1) LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupledto LDR; and (3) primary PCR coupled to secondary PCR Each of theseaspects of the invention has particular applicability in detectingcertain nucleic acid characteristics. However, each requires the use ofcoupled reactions for multiplex detection of nucleic acid sequencedifferences where oligonucleotides from an early phase of each processcontain sequences which may be used in processes used in a later phaseof the method.

Barany et al. U.S. Pat. Nos. 6,852,487, 6,797,470, 6,576,453, 6,534,293,6,506,594, 6,312,892, and 6,268,148, 6,054,564, 6,027,889, 5,830,711,5,494,810 describe the use of the ligase detection reaction (LCR) assayfor the detection of specific sequences of nucleotides in a variety ofnucleic acid samples.

Barnay et al., U.S. Pat. Nos. 7,807,431, 7,455,965, 7,429,453,7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831,7,198,894, 7,166,434, 7,097,980, 7,083,917, 7,014,994, 6,949,370,6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and6,268,148 describe the use of the ligase detection reaction (“LDR”)coupled with polymerase chain reaction (“PCR”) for nucleic aciddetection.

Barany et al., U.S. Pat. Nos. 7,556,924 and 6,858,412, describe the useof padlock probes (also called “precircle probes” or “multi-inversionprobes”) with coupled ligase detection reaction (“LDR”) and polymerasechain reaction (“PCR”) for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,709,201, and 7,198, 814describe the use of combined endonuclease cleavage and ligationreactions for the detection of nucleic acid sequences.

Willis et al., U.S. Pat. Nos. 7,700,323 and 6,858,412, describe the useof precircle probes in multiplexed nucleic acid amplification, detectionand genotyping.

Ronaghi et al., U.S. Pat. No. 7,622,281 describes amplificationtechniques for labeling and amplifying a nucleic acid using an adaptercomprising a unique primer and a barcode.

In addition to the various amplification techniques, numerous methods ofsequence determination are compatible with the assay systems of theinventions. Preferably, such methods include “next generation” methodsof sequencing. Exemplary methods for sequence determination include, butare not limited to, hybridization-based methods, such as disclosed inDrmanac, U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanacet al, U.S. patent publication 2005/0191656, which are incorporated byreference, sequencing by synthesis methods, e.g., Nyren et al, U.S. Pat.Nos. 7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos.7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc.Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing asdescribed in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311,6,828,100, and 6,210,891; and ligation-based sequencing determinationmethods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, andChurch et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

Alternatively, nucleic acid regions of interest can be selected and/oridentified using hybridization techniques. Methods for conductingpolynucleotide hybridization assays for detection of have been welldeveloped in the art. Hybridization assay procedures and conditions willvary depending on the application and are selected in accordance withthe general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred aspects. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964).

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964).

Computer Implementation of the Processes of the Invention

FIG. 1 is a block diagram illustrating an exemplary system environmentin which the processes of the present invention may be implemented forcalculating chromosome or loci dosage and fetal DNA contribution. Thesystem 10 includes a server 14 and a computer 16. The computer 16 may bein communication with the server 14 through the same or differentnetwork.

According to the exemplary embodiment, the computer 16 executes asoftware component 24 that calculates fetal chromosome dosage and/orfetal DNA contribution. In one embodiment, the computer 16 may comprisea personal computer, but the computer 16 may comprise any type ofmachine that includes at least one processor and memory.

The output of the software component 24 comprises a report 26 with avalue of probability that a locus or genomic region and/or a chromosomehas a dosage abnormality. In a preferred aspect this report is an oddsratio of a value of the likelihood that a region or chromosome has twocopies (e.g., is disomic) and a value of the likelihood that a region orchromosome has more copies (e.g., is trisomic) or less copies (e.g., ismonosomic) copies. The report 26 may be paper that is printed out, orelectronic, which may be displayed on a monitor and/or communicatedelectronically to users via e-mail, FTP, text messaging, posted on aserver, and the like.

Although the process of the invention is shown as being implemented assoftware 24, it can also be implemented as a combination of hardware andsoftware. In addition, the software 24 may be implemented as multiplecomponents operating on the same or different computers.

Both the server 14 and the computer 16 may include hardware componentsof typical computing devices (not shown), including a processor, inputdevices (e.g., keyboard, pointing device, microphone for voice commands,buttons, touchscreen, etc.), and output devices (e.g., a display device,speakers, and the like). The server 14 and computer 16 may includecomputer-readable media, e.g., memory and storage devices (e.g., flashmemory, hard drive, optical disk drive, magnetic disk drive, and thelike) containing computer instructions that implement the functionalitydisclosed when executed by the processor. The server 14 and the computer16 may further include wired or wireless network communicationinterfaces for communication.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Subjects

Subjects are enrolled upon providing informed consent under protocolsapproved by institutional review boards. Subjects are required to be atleast 18 years of age, at least 10 weeks gestational age, and to havesingleton pregnancies. A subset of enrolled subjects, consisting of 250women with disomic pregnancies, 72 women with trisomy 21 (T21)pregnancies, and 16 women with trisomy 18 (T18) pregnancies, areselected for inclusion in the study. The subjects are randomized into acohort consisting of 130 disomic pregnancies, 30 T21 pregnancies, and 9T18 pregnancies. The trisomy status of each pregnancy is confirmed byinvasive testing (fluorescent in-situ hybridization and/or karyotypeanalysis) following the analysis provided using the assay of theinvention, as described below.

Example 2: Assessment of Fetal Chromosome Dosage of Two Individual FetalChromosomes in a Maternal Sample

To assess fetal nucleic acid dosage of genomic regions of interest inthe maternal samples, assays are designed against a set of 150SNP-containing loci on each of chromosomes 21 and 18. Each assayconsists of three locus specific oligonucleotides: a left oligo with a5′ universal amplification tail, a 5′ phosphorylated middle oligo, and a5′ phosphorylated right oligo with a 3′ universal amplification tail.Two middle oligos differing by one base are used to query each SNP inthe selected loci. SNPs are optimized for minor allele frequency in theHapMap 3 dataset. Duan, et al., Bioinformation, 3(3):139-41 (2008); Epub2008 Nov. 9.

Oligonucleotides are synthesized by IDT (Coralville, Iowa) and pooledtogether to create a single multiplexed assay pool. PCR products aregenerated from each subject sample as described in U.S. Ser. No.13/013,732, filed Jan. 25, 2011; and U.S. Ser. No. 13/205,570, filedAug. 8, 2011, which are incorporated herein by reference in theirentirety. Briefly, 8 ml blood per subject is collected into a glass tubecomprising preservatives (Streck, Omaha, Nebr.) and stored at roomtemperature for up to 3 days. Plasma is isolated from blood via doublecentrifugation and stored at −20° C. for up to a year. cfDNA is isolatedfrom plasma using Viral NA DNA purification beads (Life Technologies,Carlsbad, Calif.), biotinylated, immobilized on MyOne Cl streptavidinbeads (Life Technologies, Carlsbad, Calif.), and annealed with themultiplexed oligonucleotide pool. Appropriately hybridizedoligonucleotides are catenated with Taq ligase, eluted from the cfDNA,and amplified using universal PCR primers. PCR products from 96independent samples are pooled and used as template for clusteramplification on a single lane of a TruSeq™ V3 SR flow slide (Illumina,San Diego, Calif.). The slide is processed on an Illumina HiSeg™ 2000 toproduce a 56 base locus-specific sequence and a 7 base sample tagsequence from an average of 1.18M clusters/sample.

Because the assay exhibits allele specificities exceeding 99%,informative loci are readily identified when the fetal allele dosage ofa locus is measured to be between 1 and 20%. A maximum likelihood isestimated using a binomial distribution, such as that described inco-pending application 61/509,188, filed Jul. 19, 2011, to determine themost likely fetal dosage of each of chromosome 18 and 21 based uponmeasurements from five or more informative loci. Since the likelihoodthat both chromosome 18 and 21 will exhibit a trisomy is extremely low(outside a triploid fetus), the initial risk of aneuploidy forchromosome 21 and chromosome 18 can be calculated using a computer modelthat compares the relative dosage of fetal chromosome 18 in a sample tothe relative dosage of fetal chromosome 21 in the same maternal sample.

Example 3: Aneuploidy Detection Using Comparison of Dosages of FetalChromosome 18 and Fetal Chromosome 21

The initial risk of trisomy for chromosome 18 or 21 is further optimizedusing an odds ratio that compares a model assuming a disomic fetalchromosome and a model assuming a trisomic fetal chromosome. Thedistribution of differences in observed and reference dosages areevaluated using normal distributions with a mean of 0 and standarddeviation estimated using Monte Carlo simulations that randomly drawfrom observed data. For the disomic model, p0 is used as the expectedreference dosage in the simulations. For the trisomic model, p0 isadjusted on a per sample basis with the fetal dosage adjusted referencedosage {circumflex over (P)}j, defined as

$\hat{P}j\frac{( {1 + {0.5{fj}}} ){Po}}{( {( {1 + {0.5{fj}}} ){Po}} ) + ( {1 - {Po}} )}$

where f_(i) was the fetal dosage for sample j. This adjustment accountsfor the expected increased representation of a test chromosome when thefetus is trisomic. In the simulations both p0 and f_(j) are randomlychosen from normal distributions using their mean and standard errorestimates to account for measurement variances. Simulations are executed100,000 times. The risk score is defined as the mean trisomy versusdisomy odds ratio obtained from the simulations, adjusted by multiplyingthe risk of trisomy associated with the subject's maternal andgestational age.

The risk calculation algorithm used in calculation of the estimated riskof aneuploidy uses an odds ratio comparing a mathematic model assuming adisomic fetal chromosome and a mathematic model assuming a trisomicfetal chromosome.

When X_(j)=P_(j)−P_(o) is used to describe the difference of theobserved dosage P_(j) for sample j and the estimated reference dosageP_(o), the risk calculation algorithm used computed:

$\frac{P( {{xj}\mspace{14mu} 1\; T} )}{P( {{xj}\mspace{14mu} 1\; D} )}$

where T was the trisomic model and D was the disomic model. The disomicmodel D was a normal distribution with mean 0 and a sample specificstandard deviation estimated by Monte Carlo simulations as describedbelow. The trisomic model T was also a normal distribution with mean 0,determined by transforming x_(j) to x_(j)=p_(j)−{circumflex over(P)}_(j), the difference between the observed dosage and a fetalfraction adjusted reference dosage as defined by:

$\hat{P}j\frac{( {1 + {0.5{fj}}} ){Po}}{( {( {1 + {0.5{fj}}} ){Po}} ) + ( {1 - {Po}} )}$

where f was the fetal fraction for sample j. This adjustment accountedfor the expected increased representation of a trisomic fetalchromosome. Monte Carlo simulations were used to estimate samplespecific standard deviations for disomic and trisomic models of dosagedifferences. Observed dosages for each sample were simulated bynon-parametric bootstrap sampling of loci and calculating means, orparametric sampling from a normal distribution using the mean andstandard error estimates for each chromosome from the observednon-polymorphic locus counts. Similarly, the reference dosage p0 andfetal fraction f were simulated by non-parametric sampling of samplesand polymorphic loci respectively, or chosen from normal distributionsusing their mean and standard error estimates to account for measurementvariances. Parametric sampling was used in this study. Simulations wereexecuted 100,000 times, and dosage differences were computed for eachexecution to construct the distributions. Based on the results of thesesimulations, normal distributions were found to be good models of disomyand trisomy.

The final risk calculation algorithm risk score is defined as

$\frac{{P( {{xj}\mspace{14mu} 1\; T} )}{P(T)}}{{P( {{xj}\mspace{14mu} 1\; D} )}{P(D)}}$

where P(T) I P(D) is the prior risk of trisomy vs. disomy. The data onprior risk of aneuploidy was taken from well-established tablescapturing the risk of trisomy associated with the subject's maternal andgestational age (Nicolaides K H. Screening for chromosomal defects.Ultrasound Obstet Gynecol 2003; 21:313-321).

Example 4: Assessment of Fetal Chromosome Dosage of One Individual FetalChromosome and Two or More Comparative Chromosomes in a Maternal Sample

Assays are designed against a set of 20 SNP-containing loci onchromosome X outside the pseudoautosomal region, 20 SNP-containing locion chromosome X within the pseudoautosomal region, and 20 SNP-containingloci distributed amongst chromosomes 1-10. Each assay consists of threelocus specific oligonucleotides: a left oligo with a 5′ universalamplification tail, a 5′ phosphorylated middle oligo, and a 5′phosphorylated right oligo with a 3′ universal amplification tail. Twomiddle oligos differing by one base are used to query each SNP in theselected loci. SNPs are optimized for minor allele frequency in theHapMap 3 dataset. Duan, et al., Bioinformation, 3(3):139-41 (2008); Epub2008 Nov. 9.

Oligonucleotides are synthesized by IDT (Coralville, Iowa) and pooledtogether to create a single multiplexed assay pool. PCR products aregenerated from each subject sample as described in U.S. Ser. No.13/013,732, filed Jan. 25, 2011; and U.S. Ser. No. 13/205,570, filedAug. 8, 2011. Briefly, 8 ml blood per subject is collected into a glasstube comprising preservatives (Streck, Omaha, Nebr.) and stored at roomtemperature for up to 3 days. Plasma is isolated from blood via doublecentrifugation and stored at −20° C. for up to a year. cfDNA is isolatedfrom plasma using Viral NA DNA purification beads (Life Technologies,Carlsbad, Calif.), biotinylated, immobilized on MyOne Cl streptavidinbeads (Life Technologies, Carlsbad, Calif.), and annealed with themultiplexed oligonucleotide pool. Appropriately hybridizedoligonucleotides are catenated with Taq ligase, eluted from the cfDNA,and amplified using universal PCR primers. PCR products from 96independent samples are pooled and used as template for clusteramplification on a single lane of a TruSeq™ V3 SR flow slide (Illumina,San Diego, Calif.). The slide is processed on an Illumina HiSeg™ 2000 toproduce a 56 base locus-specific sequence and a 7 base sample tagsequence from an average of 1.18M clusters/sample.

A maximum likelihood is estimated using a binomial distribution, such asthat described in co-pending application 61/509,188, filed Aug. 8, 2011to determine the most likely fetal dosage of chromosome X and collectivefetal dosage of non-aneuploid chromosomes 1-10 based upon measurementsfrom five or more informative loci. Since chromosomes 1-10 are notexpected to exhibit any evidence of aneuploidy, the fetal concentrationcalculated across these chromosomes can be used as a direct comparatorwith the calculated contribution of chromosome X for determining therisk of either monosomy or trisomy of chromosome X.

The presence of trisomy X can be determined by a direct comparison ofthe contribution of the fetal X as determined inside and/or outside thepseudoautosomal regions compared to the fetal contribution calculatedfrom the collective data of fetal chromosome 1-10.

The presence of monosomy X requires distinguishing an XO monosomygenotype from the presence of an XY normal genotype. The initialdetermination of monosomy can be calculated by a comparison of thecontribution of the fetal X as determined outside the pseudoautosomalregion. The genotype is then further distinguished by comparison to thefetal X contribution inside the pseudoautosomal regions which are incommon with the Y chromosome, or by combining the assay with detectionof a Y sequence in the maternal sample. The disomic levels of fetal X asdetermined inside the pseudoautosomal region or other detection of thepresence of Y combined with a determination of the fetal X monosomy isindicative of a male genotype rather than XO.

While this invention is satisfied by aspects in many different forms, asdescribed in detail in connection with preferred aspects of theinvention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific aspects illustrated anddescribed herein. Numerous variations may be made by persons skilled inthe art without departure from the spirit of the invention. The scope ofthe invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, 916.

1-20. (canceled)
 21. A computer-implemented process to calculate a riskof a fetal aneuploidy in a maternal serum or plasma sample from apregnant female, the computer-implemented process comprising thefollowing steps: 1) estimating the chromosome dosage of a first fetalchromosome in the maternal sample; 2) estimating the chromosome dosageof one or more other fetal chromosomes in the maternal sample; 3)providing data on prior risk of aneuploidy for at least the first fetalchromosome based on extrinsic characteristics; 4) calculating a value ofthe likelihood that a first fetal chromosome is aneuploid by comparingthe chromosome dosage of the first fetal chromosome to the chromosomedosage of the one or more other fetal chromosomes in view of the priorrisk of aneuploidy; 5) calculating a value of the likelihood that thefirst fetal chromosome is disomic by comparing the chromosome dosage ofthe first fetal chromosome to the chromosome dosage of the one or moreother fetal chromosomes in view of the prior risk of aneuploidy; and 6)calculating a risk of aneuploidy of the first fetal chromosome based onthe calculated values of likelihood.
 22. (canceled)
 23. (canceled) 24.The process of claim 21, wherein the maternal sample comprises cells.25. The process of claim 21, wherein the data on prior risk ofaneuploidy comprises information related to maternal age.
 26. Theprocess of claim 21, wherein the data on prior risk of aneuploidycomprises information related to gestational age.
 27. The process ofclaim 21, wherein the chromosome dosage of the first chromosome isestimated interrogating at least twenty polymorphic loci on the firstchromosome.
 28. The process of claim 27, wherein the chromosome dosageof the first chromosome is estimated by interrogating at least fiftypolymorphic loci on the first chromosome.
 29. The process of claim 21,wherein the chromosome dosage of the first chromosome is estimated byinterrogating at least five informative loci on the first chromosome.30. The process of claim 29, wherein the chromosome dosage of the firstchromosome is estimated by interrogating at least twenty informativeloci on the first chromosome.
 31. The process of claim 21, wherein thevalue of the probability of an aneuploidy is an odds ratio.
 32. Theprocess of claim 21, wherein the value of the probability of ananeuploidy for the first fetal chromosome is based on a value of thelikelihood of the chromosome being trisomic and a value of thelikelihood of the chromosome being disomic.
 33. The process of claim 21,wherein the value of the probability of a chromosome dosage abnormalityfor the first fetal chromosome is based on a value of the likelihood ofthe chromosome being monosomic and a value of the likelihood of thechromosome being disomic.
 34. The process of claim 21, furthercomprising the following step: interrogating at least twenty polymorphicloci on a first fetal chromosome in the maternal sample andinterrogating at least twenty polymorphic on a second fetal chromosomein the maternal sample.
 35. The process of claim 34, further comprisingthe following step: determining fetal DNA contribution to the maternalsample.
 36. The process of claim 35, wherein the calculated values oflikelihood are determined in view of the fetal DNA contribution to thematernal sample.